Cellular damage caused by oxidative stress is central to the pathology of a wide variety of diseases. Mammalian cells defend against oxidative stress through a conserved transcriptional response in which the Nrf proteins induce expression of Phase II detoxification enzyme genes. Much remains to be learned about how this oxidative stress defense is controlled, and might be harnessed therapeutically. In the nematode C. elegans, we have shown that this oxidative stress response is orchestrated by the SKN-1 transcription factor, which is related to Nrf proteins. In the embryo, maternally provided SKN-1 initiates development of the feeding/digestive system. We have found that during postembryonic stages SKN-1 is expressed in the intestine and ASI chemosensory neurons, and is required for resistance to oxidative stress and for normal longevity. We have also obtained the novel and exciting observation that in the intestine SKN-1 localization is regulated by p38, GSK-3, and insulin-like signaling, apparently through direct SKN-1 phosphorylation. Our findings indicate that SKN-1 integrates multiple stress and metabolic inputs, and provide a valuable whole-organism model for studying this stress response. In the new project, we will investigate how these signals regulate SKN-1 and its functions, and employ advantages of C. elegans to identify additional mechanisms that control this stress response. In Aim 1, we will determine how expression of SKN-1 in different tissues contributes to its functions, and we will test models for how SKN-1 is regulated in the intestine, including the exciting hypothesis that one SKN-1 form responds to mitochondrial stress. In Aim 2 we will study how insulin-like signaling regulates SKN-1, and how SKN-1 influences biological effects of insulin-like signaling. In Aim 3, we will use microarrays to identify additional SKN-1 target genes and functions, and RNA interference screening to identify novel mechanisms that regulate SKN-1. This work will greatly expand our understanding of how this oxidative stress response is regulated, and of the biological functions of the protein that orchestrates this response. Lay Summary: Oxidative stress (excessive levels of cellular free radicals) is important in many diseases, including diabetes, atherosclerosis, and cancer. A new means of combating oxidative stress would be to harness innate cellular mechanisms that can defend against it. In this project, we will use a simple nematode model to identify and study mechanisms that control this stress defense, and to investigate how this stress defense contributes to important biological processes that include responses to insulin.